Double bands in DNA gel electrophoresis caused by bis-intercalating dyes

Many bis-intercalating dyes used for fluorescence detection of DNA in electrophoresis have been reported to give band-splitting and band-broadening, which results in poor resolution and a decreased detection sensitivity. We have studied the dimeric dye YOYO-1, and to some extent also TOTO-1 and EthD-1, and found that in complex with DNA these dyes give rise to two components with different electrophoretic mobilities. Electrophoresis experiments and spectroscopic measurements on the two components show that they differ in that the DNA molecules have different amounts of dye bound. Our results exclude that the extra bands are caused by intermolecular cross-linking. Incubation of the samples for increasing times before electrophoresis makes the bands move closer and closer to each other as the dye molecules become more homogeneously distributed among the DNA molecules. Finally, the two bands merge into one at an intermediate position. This equilibration process is extremely slow at room temperature (days), and is therefore not a practical method to eliminate band-splitting in routine analysis. However, we find that if the temperature is raised to 50 degrees C, the dye-DNA complexes equilibrate completely in only 2 h.